Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. We have recently shown that the GTPase dynamin, best studied for its role in membrane fission, also regulates early stages of CME in an unexpectedly isoform-specific manner. We have found that dynamin-1 (Dyn1), previously considered the neuron-specific isoform, is in fact ubiquitously expressed, but regulated by phosphorylation in nonneuronal cells. Dyn1 is upregulated in many cancer cells, including nonsmall cell lung cancer (NSCLC) cells and activated downstream of the oncogenic kinase, Akt. Activation of Dyn1 in nonneuronal cells accelerates CME by increasing the rates of clathrin coated pit (CCP) initiation and maturation. The Dyn1- dependent alterations in CME enhance signaling downstream of EGFR. We also discovered that Dyn1, but not Dyn2, is required for CME of TRAIL-activated death receptors (DRs), whereas in the same cells Dyn2, but not Dyn1 is required for CME of constitutively-internalized transferrin receptors. Dyn1-dependent endocytosis suppresses apoptotic signaling downstream of TRAIL-activated DRs. Based on these new findings, we hypothesize that the GTPase Dyn1 functions, in an isoform-specific manner, as a nexus controlling the reciprocal regulation of cell surface receptor signaling and CME. Our discoveries raise many new questions, including: How is Dyn1 activity regulated in nonneuronal cells? What are the biochemical properties of Dyn1 vs Dyn2 that are essential for its isoform-specific early roles in CCP initiation and maturation? What are the upstream and downstream effectors and mechanisms responsible for the profound, Dyn1 isoform- specific effects on CME and signaling? To address these questions, in Aim 1 we will functionally analyze Dyn1/Dyn2 chimeras to identify the domain(s) and biochemical properties required for Dyn1 isoform-specific activities and identify the phosphorylation events and kinases/phosphatases that regulate Dyn1 in nonneuronal cells. In Aim 2 we will take a quantitative proteomics approach to identify and then functionally validate active Dyn1-specific upstream and downstream effectors. Finally, studies of dynamin function in living cells would be greatly facilitated by small molecule inhibitors, yet the efficacy and specificity of current dynamin inhibitors is in doubt. Therefore, using a new, highly sensitive and high-throughput compatible assay for dynamin GTPase activity, in Aim 3 we will develop potent and specific small molecule inhibitor(s) of dynamin as a tool to study its role(s) in regulating CME and signaling. Our new findings linking Dyn1 function to the reciprocal regulation of signaling and CME provide us with a molecular handle to begin to probe the `outside-in' regulation of CME and signaling. Results from these studies are significant as these regulatory loops affect intracellular signaling, and hence many physiological processes critical in human health and disease.